The overall objective of this proposal is to develop a clinical-grade Megakaryocyte Development Medium (MDM) that supports the ex vivo development of human megakaryocytes. Use of this unique medium will provide information on several critical gap-areas concerning these elusive yet critically important cells; lipid metabolism, regulation of maturation, and clinical utility. The innovative aspects are the studies on enhancing lipid metabolism, the use of cytokine/matrix molecule combinations, and an in vivo megakaryocyte-transplantation model. MDM will have clinical use in developing therapies for thrombocytopenia and other platelet disorders, in that it allows generation of large numbers of megakaryocytes (> 10/9) that are not possible via routine bone marrow aspiration. MDM also allows for the standardization of megakaryocyte culture conditions between laboratories. Building on the successful results of our Phase I studies, we have designed relevant and highly critical experiments in our Phase II proposal to investigate the following goals. Specific Aim 1 addresses the further optimization of MDM, with a particular focus on enhancing megakaryocyte lipid metabolism. Specific Aim 2 examines the effects of specific cytokines and matrix molecules as complex regulatory signals. Specific Aim 3 is to test and/or long-term engraftment. This aim also provides a pre-clinical animal model for cellular-based therapies of platelet disorders. Specific Aim 4 is to scale these cultures up to levels producing 1-5 x 10/9 megakaryocytes per culture. Successful completion of this Phase II proposal will result in a widely used commercial product, a clinically relevant animal model, and much needed information to further our studies on the development of these biologically important cells. PROPOSED COMMERCIAL APPLICATION: The development of clinical grade reagents that will support megakaryocyte proliferation and differentiation will be valuable in developing novel approaches for the treatment of hematopoietic disorders such as thrombocytopenia.